Palliative Role of Zamzam Water against Cyclosporine-Induced Nephrotoxicity through Modulating Autophagy and Apoptosis Crosstalk

Cyclosporine (CsA) is considered one of the main components of treatment protocols for organ transplantation owing to its immunosuppressive effect. However, its use is very restricted due to its nephrotoxic effect. ZW is an alkaline fluid rich in various trace elements and has a great ability to stimulate antioxidant processes. This study aimed to investigate the possible mitigating effect of ZW on CsA-induced nephrotoxicity and its underlying mechanisms. Forty rats were allocated into four groups (n = 10): a control group, ZW group, cyclosporine A group (injected subcutaneously (SC) with CsA (20 mg/kg/day)), and cyclosporine A+ Zamzam water group (administered CsA (SC) and ZW as their only drinking water (100 mL/cage/day) for 21 days). Exposure to CsA significantly (p < 0.001) increased the serum creatinine level, lipid peroxidation marker level (malondialdehyde; MDA), and the expression of apoptotic markers procaspase-8, caspase-8, caspase- 9, calpain, cytochrome c, caspas-3, P62, and mTOR in renal tissues. Meanwhile, it markedly decreased (p< 0.001) the autophagic markers (AMPK, ULK-I, ATag5, LC3, and Beclin-1), antiapoptotic Bcl-2, and antioxidant enzymes. Moreover, the administration of CsA caused histological alterations in renal tissues. ZW significantly (p < 0.001) reversed all the changes caused by CsA and conclusively achieved a positive outcome in restraining CsA-induced nephrotoxicity, as indicated by the restoration of the histological architecture, improvement of renal function, inhibition of apoptosis, and enhancement of autophagy via the AMPK/mTOR pathway.


Introduction
Cyclosporine A (CsA), a natural cyclic peptide extracted from the fungus Beauveria nivea, is considered as one of the most important components of prophylaxis treatment protocols for organ transplantation and autoimmune diseases [1]. It inactivates T lymphocytes through the inhibition of the calcineurin enzyme via its attachment to the cytosolic In this study, 40 rats weighing 200-250 g were obtained from the Medical Experimental Research Center in Mansoura University. These animals were kept in the Pharmacology Department of the Faculty of Veterinary Medicine in Mansoura University under standardized conditions at 24 ± 2 • C and a 12 h light-dark cycle. The rats were offered food and water ad libitum and were allowed to acclimate one week prior to the onset of the experiment. The Mansoura University Animal Care and Use Committee (MU-ACUC) endorsed the animal experiment (approval no. VM.R. 22.10.20).

Chemicals
Cyclosporine (Sandimmune ® , 50 mg/mL ampule) was purchased from Novartis (Istanbul, Turkey). ZW was brought from the Zamzam well located within the holy mosque in Makkah, Saudi Arabia.

Experimental Design
The rats were divided into four groups (10 rats/group): a control group, in which rats received oral 0.9% saline at a rate of 0.3 mL/rat and tap water for 21 days; a ZW group, in which rats received ZW (100 mL/cage/day) as their only supply of drinking water for 21 days.; a CsA group, injected subcutaneously (SC) with CsA at 20 mg/kg/day and given oral 0.9% saline at a rate of 0.3 mL/rat for 21 days; and a CsA+ ZW group, wherein rats received an SC injection of cyclosporine at 20 mg/kg daily and were provided with ZW (100 mL/cage/day) as their only supply of drinking water for 21 days. The CsA dose used in this experiment was selected based on previous research published by Ateyya [20], El-Sheikh et al. [21], Abdel-latif et al. [22], and Mohamadin et al. [23], who reported that treatment with CsA at this dose causes severe nephrotoxicity. The dose of ZW used in this experiment was based on the study performed by Mraisel and Abu Ali, [24], who investigated its association in relation to kidney damage in rats.

Specimen Collection
The rats received anesthesia with sodium thiopental intraperitoneally at 50 mg/kg on day 21. The serum was obtained from blood harvested from the retroorbital venous plexus through a capillary tube. The rats were sacrificed via cervical dislocation. The kidneys were excised and washed with saline. The serum and portions of the kidneys were kept at −80 • C for further biochemical and molecular investigations. In addition, other parts of renal tissues were fixed in 10% formalin and then embedded in a paraffin block for histological and immunohistochemical assessment. The remaining kidney specimens were homogenized with a radioimmunoprecipitation assay buffer (RIPA buffer) completed with a cocktail of protease inhibitors, and the homogenate was then stored for 15 min at 4 • C. Following this, it was centrifuged at 15,000 g at 4 • C, and the resulting supernatant was stored at −80 • C for Western blotting assessment.

Renal Function Parameters
Blood urea nitrogen (BUN) and serum creatinine were assessed following the methods described by Kumar et al. [25] and Piéroni et al. [26], respectively. Additionally, creatinine clearance (Cr Cl) was estimated as follows: Cr Cl(mL/min) = Urine volume mL 24 h × Urine creatininecon cnetration mg dL plasma creatinine mg dL

Assessment of Oxidative Stress Biomarkers
Renal tissues were assessed for the lipid peroxidation marker malonaldehyde (MDA) with the use of commercial kits (Cat# No. MD [25][26][27][28][29] and the colorimetric method at 534 nm following the method of Mesbah et al. [27]. Furthermore, superoxide dismutase (SOD) activity was examined with colorimetry at 560 nm using commercial kits (Cat# No. SD25-21) as described by Xu et al. [28]. For the assessment of glutathione peroxidase (GPx), commercial kits (Cat# GP 2524) were used according to the instructions reported by Zhang et al. [29]. The activity of catalase (CAT) was investigated as described by Baureder et al. [30] (Cat# Ca 2517). All the kits were supplied by Biodiagnostics Co (Cairo, Egypt).

Histopathological and Immunohistochemical Examination
After fixation of the kidney in 10% formalin and its immersion in a paraffin block, 5 µm thick sections of the paraffin block were prepared for staining with hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) stains. The kidney damage was evaluated and scored according to the following changes in the renal corpuscle: Bowman's capsule and space widening, shrinkage of the glomeruli, nuclear pyknosis, and edema of the interstitial tissue and perivascular space. Another change observed was the alterations recorded in the renal tubules, which may come in the form of hyaline casts inside the renal tubular lumens. The scores were documented as published by Zahran et al. [31]. Five random fields for each slide were used for scoring. A score of 0 represented no damage, while a score of 1 indicated 10% damage, that of 2 indicated 11-25% damage, a score of 3 represented 26-45% damage, a score of 4 represented 46-75% damage, and a score of 5 indicated more than 76% damage. For the immunohistochemical examination of the renal tissues, 5 µm thick sections were deparaffined using xylene with the descending scale of ethanol for dehydration. To retrieve the antigen, sections of the renal tissues were immersed inside a hot buffer (citrate) for 10 min, and then 0.3% H 2 O 2 was applied. Thereafter, the sections were incubated with the primary antibodies of LC3 (Cat# GB13431 1:300), P62 (Cat# A19700 1:200), Beclin 1 (Cat# GB11228 1:500), Caspase 3 (Cat# GB11532 1:500), Bax (Cat# A12009 1:100), and Bcl-2 (Cat# Clone 124 1:200). Following this, the enzyme horseradish peroxidase (HRP) (Dako C., Denmark) and secondary antibody (goat antirabbit, Cat# K205587, EN vision) were incubated at 25 • C for half an hour. Sections were mounted with 3,3 diaminobenzidine and then counter-stained with hematoxylin. The slides were examined by histopathologists at the Department of Pathology, Faculty of Veterinary Medicine, Mansoura University, Egypt.

Analysis of Apoptosis with Flow Cytometry
The primary proximal tubular epithelial cells (PTECs) from different experimental groups were isolated according to the previous method performed by Liu et al. [33]. The cells were maintained in K1 culture medium DMEM/F12 + GlutaMax (Invitrogen GmbH); supplemented with 5% FBS (PAN-Biotech GmbH; Aidenbach, Germany), 1% penicillin/streptomycin, insulin (5 µg/mL), hydrocortisone (1.8 µg/mL), transferrin (5 µg/mL), sodium selenite (173 ng/mL), murine epidermal growth factor (25 ng/mL), and triiodothyronine (6.5 ng/mL); and then seeded into collagen R-coated (20 µg/mL) 6-well culture plates (TPP AG, Trasadingen, Switzerland) at a density of approximately 1 × 105 cells per well. The cells were incubated with 5% CO 2 at 37 • C, in a similar fashion to that conducted with the rat cell line NRK52E, for 24 h. The cells were collected in free trypsin-EDTA, centrifuged for 5 min at 1000 rpm, and then washed with PBS. After being resuspended in the binding buffer, the cells were treated with the use of Annexin V−FITIC and PI apoptosis detection kits (KEYGen BioTech, Nanjing, China) for 15 min and were subsequently analyzed with flow cytometry (Becton Dickinson). Channel FL1 was used to detect Annexin V−FITIC green fluorescence for the test, and channel PI was used to detect red fluorescence (FL3). The Accuri C6 package was used to calculate the Annexin V−FITIC and PI-stained cells.

RT-PCR Assessment
Total RNA was isolated from the tissue homogenate of the different groups using Direct-Zol RNA Miniprep plus (Cat# R2070. ZYMO research corporation, Irvine, CA USA). A Beckmann Daul spectrophotometer (USA) was used for quantitative and qualitative determination. Thermos Fisher Scientific (Waltham, MA, USA) provided the SuperScript IV One-Step RT-PCR kit (Cat# 12594100) for the determination of the reverse transcriptase and PCR of the isolated RNA. Using a 96-well Step One instrument (Applied biosystems, Foster City, CA, USA), thermal profiling was performed as follows: reverse transcription for 10 min at 45 • C; RT inactivation for 2 min at 98 • C; and initial denaturation for 40 cycles of 10 s at 98 • C, 10 s at 55 • C, and 30 s at 72 • C. Real-time PCR results for the housekeeping and target genes are expressed in cycle threshold. The 2-∆∆Ct method was used to determine the mean critical threshold expression value of the GAPDH housekeeping gene and the target gene in order to minimize the expression variation. Primer sequences for the AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and glyceraldehyde3. phosphate (GAPDH) genes are listed in Table 1.

Analysis of Zamzam Water
A total of 300 mL of ZW was obtained from Al Haram pipes using a 500 mL glass bottle. The analysis was completed in 24 h using atomic absorption spectrophotometry measurements from the SETE Saudia Building Quality for life (ALFATAH WATER AND POWER) company, Jeddah, Saudi Arabia.

Statistical Analysis
The data were analyzed using the Graph Pad Prism program version 8.0 (La Jolla, CA, USA). The values of different groups are presented as mean ± SD, and a comparison of the different groups was carried out with one-way ANOVA, followed by the Tukey-Kramer post hoc test. p values less than 0.05 were regarded as significant.

Effect of Zamzam Water on CsA A-Induced Renal Impairment
The rats that received CsA revealed remarkable renal impairment as compared to the control group, which was evidenced by the significant elevation (p < 0.001) of the serum level of creatinine and BUN, with a marked reduction (p < 0.001) in creatinine clearance. Interestingly, drinking ZW with CsA injection significantly (p < 0.001) improved renal function by decreasing creatinine and BUN serum levels as well as increasing creatinine clearance relative to the CsA group (Table 2); there was no significant difference between the control group and ZW group.

Effect of Zamzam Water against CsA-Induced Oxidative Stress
Rats inoculated with CsA exhibited a significant (p < 0.001) elevation of the lipid peroxidation marker (MDA) level and a reduction in the activities of antioxidant enzymes SOD, CAT, and GPx in renal tissues as compared to control rats. ZW, with its powerful antioxidant character, markedly (p < 0.001) decreased the homogenate level of MDA and enhanced the activities of SOD, CAT, and GPx in comparison with the CsA group (Table 3). There were no significant differences between the control group and ZW group despite the increase in antioxidative stress markers in the ZW group. Data are presented as the mean ± SD. One-way ANOVA with Tukey's analysis; *** p < 0.001 vs. control; ### p < 0.001 vs. CsA. MDA-malondialdehyde; SOD-superoxide dismutase; CAT-catalase; GPx-glutathione peroxidase.

Ameliorative Impact of Zamzam Water on Renal Morphology
A histopathological investigation of the kidneys of the control and ZW groups showed normal tubules and glomeruli ( Figure 1A-D). On the contrary, treatment with CsA revealed significant (p < 0.001) alternations in the renal architecture in the form of diffuse necrosis in the cortical cells, with the presence of hypereosinophilic cytoplasm and nuclear pyknosis with large intraluminal sloughed tubular epithelial cells and necrotic debris (granular casts) as well as proliferative glomerular capillaries ( Figure 1E,G). Furthermore, medullary tubules showed diffuse medullary tubular necrosis and vacuolation with focal interstitial fibrosis ( Figure 1F,H). Drinking ZW was associated with a marked histological improvement in the form of occasional individual tubular necrosis with diffuse minimal-to-mild tubular interstitial congestion ( Figure 1I,J). Additionally, CsA administration was associated with marked glycogen deposition in the form of PAS-positive staining in dilated cortical and medullary tubules with intraluminal proteinaceous globules ( Figure 2E,F). In contrast, the CsA+ ZW group showed a significant decrease (p < 0.001) in glycogen deposition in the form of focal intracytoplasmic-positive PAS in the tubular epithelium of the cortex and a positive staining of the tubular basement membrane of medullary tubules ( Figure 2G,H). This improvement in the morphological structures of the renal glomerulus and tubules explains the ameliorative effect of ZW on renal function. staining in dilated cortical and medullary tubules with intraluminal proteinaceous globules ( Figure 2E,F). In contrast, the CsA+ ZW group showed a significant decrease (p < 0.001) in glycogen deposition in the form of focal intracytoplasmic-positive PAS in the tubular epithelium of the cortex and a positive staining of the tubular basement membrane of medullary tubules ( Figure 2G,H). This improvement in the morphological structures of the renal glomerulus and tubules explains the ameliorative effect of ZW on renal function.  group shows diffuse necrotic cortical tubules with a large intraluminal hyaline cast. (H) Medullary tubular sloughing (thin arrow) and intraluminal hyaline cast (star) with interstitial congestion (thick arrow). (I) The CsA + ZW group displays occasional individual tubular necrosis (thin arrow). (J) The CsA + ZW group exhibits diffuse minimal-to-mild interstitial congestion (thick arrow). K) Histogram of the percentage area of the histological score. *** p < 0.001 vs. control; ### p < 0.001 vs. CsA. Image magnification = 400×; scale bar = 50 µm.

Effect of Zamzam Water against CsA-Induced Renal Tubular Apoptosis
The subcutaneous injection of CsA induced a substantial (p< 0.001) rise in the immunoexpression of the proapoptotic protein Bax ( Figure 3E,F) and the number of renal tubular apoptotic caspase-3 cells ( Figure 4E,F). Additionally, it increased the protein expression of the apoptotic proteins caspase-3, procaspase-8, caspase-8, caspase-9, cytochrome

Effect of Zamzam Water against CsA-Induced Renal Tubular Apoptosis
The subcutaneous injection of CsA induced a substantial (p< 0.001) rise in the immunoexpression of the proapoptotic protein Bax ( Figure 3E,F) and the number of renal tubular apoptotic caspase-3 cells ( Figure 4E,F). Additionally, it increased the protein expression of the apoptotic proteins caspase-3, procaspase-8, caspase-8, caspase-9, cytochrome c, and calpain, as indicated by the Western blot assay ( Figure 5). It also significantly (p < 0.001) decreased the immunoexpression of Bcl-2 ( Figure 6E,F) in comparison to the control group. Interestingly, the concomitant administration of ZW and CsA significantly (p < 0.001) antagonized their apoptotic effect on renal tubular epithelial cells by decreasing the immunoexpression of Bax ( Figure 3G,H) and the Western blotting protein expression of caspase-3, procaspase-8, caspase-8, caspase-9, cytochrome c, and calpain ( Figure 5). Moreover, ZW upregulated the immunoexpression of Bcl-2 ( Figure 6G,H). With regard to the flow cytometric examination of in vitro Annexin-V-labeled renal tubular apoptotic cells, CsA significantly (p < 0.001) increased the level of the labeled apoptotic cells, which counterweighed the control group ( Figure 7C). Interestingly, ZW administration significantly (p < 0.001) decreased the count of apoptotic cells in comparison to the CsA group. ZW exhibited a cytoprotective function in response to CsA-induced renal tubular apoptosis by inhibiting the extrinsic apoptotic pathway proteins procaspase-8 and caspase-8 as well as the intrinsic pathway proapoptotic protein Bax, mitochondrial membrane protein cytochrome c, and caspase-9 protein. Subsequently, there was a decrease in the percentage of cleaved caspase-3 apoptotic cells with the activation of the antiapoptotic protein Bcl-2.

Effect of Zamzam Water on Autophagic Magnitude in the Kidney Epithelial Cells of Rats with CsA-Induced Nephrotoxicity
In comparison to the control group, CsA administration downregulated autophagic renal tubular epithelial cell death by significantly (p < 0.001) decreasing the immunoexpression of the autophagy regulator protein Beclin-1 and the autophagosome membrane protein LC3 II ( Figures 8E,F and 9E,F), with the downregulation of the protein level of autophagy-related protein ATg5. This was elucidated with Western blot analysis ( Figure  11C) and the upregulation of the immunoexpression of P62 ( Figure 10E,F), which is considered to be an indicator of the inhibition of the autophagic process. On the other hand, ZW counteracted the negative effect of CsA on the autophagy flux by the significant (p < 0.001) upregulation of Beclin-1 and LC3 II immunoexpression and the protein level of Atg5, which was concomitant with the downregulation of P62 immunoexpression in relation to the CsA group. This indicates that ZW improved cellular homeostasis by promoting autophagic renal tubular epithelial cell death and enhancing cellular survival in response to cellular apoptosis shift.

Effect of Zamzam Water on Autophagic Magnitude in the Kidney Epithelial Cells of Rats with CsA-Induced Nephrotoxicity
In comparison to the control group, CsA administration downregulated autophagic renal tubular epithelial cell death by significantly (p < 0.001) decreasing the immunoexpression of the autophagy regulator protein Beclin-1 and the autophagosome membrane protein LC3 II ( Figure 8E,F and Figure 9E,F), with the downregulation of the protein level of autophagy-related protein ATg5. This was elucidated with Western blot analysis ( Figure 10C) and the upregulation of the immunoexpression of P62 ( Figure 11E,F), which is considered to be an indicator of the inhibition of the autophagic process. On the other hand, ZW counteracted the negative effect of CsA on the autophagy flux by the significant (p < 0.001) upregulation of Beclin-1 and LC3 II immunoexpression and the protein level of Atg5, which was concomitant with the downregulation of P62 immunoexpression in relation to the CsA group. This indicates that ZW improved cellular homeostasis by promoting autophagic renal tubular epithelial cell death and enhancing cellular survival in response to cellular apoptosis shift.

Effect of Zamzam Water on the AMPK/mTOR Pathway
In order to elucidate the positive effect of ZW on the autophagic process, its effect on the AMPK/mTOR pathway, which is considered to be the main regulatory pathway in the autophagy process, was examined. The subcutaneous injection of CsA significantly (p < 0.001) inhibited the AMPK-ULK-1 interaction by downregulating the phosphorylation of AMPK (Figures 11D and 12A) and ULK-1 ( Figure 11B) at the level of gene expression for AMPK and at the protein level for AMPK and ULK-1. This is considered to be a positive pathway for the induction of the autophagic process during CsA-induced nephrotoxicity. Furthermore, the phosphorylation of mTOR ( Figures 11E and 12B), a negative regulator of autophagy, was upregulated in CsA group as compared to the control group. Interestingly, drinking of ZW significantly (p < 0.001) upregulated AMPK at the mRNA and protein levels while upregulating ULK-1 at the protein level; mTOR was also downregulated at the mRNA and protein levels in CsA+ZW group in comparison with the CsA group. The regulatory effect of ZW on the AMPK/mTOR pathway indicated its positive effect on the autophagy process.

Effect of Zamzam Water on the AMPK/mTOR Pathway
In order to elucidate the positive effect of ZW on the autophagic process, its effect on the AMPK/mTOR pathway, which is considered to be the main regulatory pathway in the autophagy process, was examined. The subcutaneous injection of CsA significantly (p < 0.001) inhibited the AMPK-ULK-1 interaction by downregulating the phosphorylation of AMPK (Figures 10D and 12A) and ULK-1 ( Figure 10B) at the level of gene expression for AMPK and at the protein level for AMPK and ULK-1. This is considered to be a positive pathway for the induction of the autophagic process during CsA-induced nephrotoxicity. Furthermore, the phosphorylation of mTOR ( Figures 10E and 12B), a negative regulator of autophagy, was upregulated in CsA group as compared to the control group. Interestingly, drinking of ZW significantly (p < 0.001) upregulated AMPK at the mRNA and protein levels while upregulating ULK-1 at the protein level; mTOR was also downregulated at the mRNA and protein levels in CsA+ZW group in comparison with the CsA group. The regulatory effect of ZW on the AMPK/mTOR pathway indicated its positive effect on the autophagy process.

Discussion
Cyclosporine A administration in organ transplantation is mandatory owing to its immunosuppressive properties, but its prolonged usage causes many side effects, one of which is oxidative-stress-induced nephrotoxicity [34][35][36]. It has been reported that antioxidant intake positively protects individuals against oxidative-insult-induced nephrotoxicity [34]. The antioxidant power of ZW is highly regarded for its alkaline ionized nature [20,37,38]. Analyses of ZW have revealed its high amounts of beneficial trace elements such as zinc, Mg, and selenium, which give ZW its antioxidant and antiapoptotic character [39,40]. Therefore, this study investigated the possible protective effect of ZW against CsA-induced nephrotoxicity along with its underlying mechanisms. From our findings, we can conclude that ZW improves renal impairment induced by CsA administration by decreasing creatinine and BUN levels and elevating creatinine clearance. Moreover, drinking ZW combats CsA-induced oxidative stress by decreasing MDA, a lipid oxidation marker, and enhancing the activities of the antioxidant enzymes SOD, GPx, and CAT, resulting in a marked improvement in the renal architecture. Additionally, ZW eliminates renal tubular epithelial cell apoptosis via a decrease in the apoptotic markers procaspase-8, caspase-8, case-9, caspase-3, cytochrome c, and BAX along with the augmentation of antiapoptotic Bcl-2 immunoexpression as well as the upregulation of the autophagy markers Beclin-1, LC3, and Atg5 and the downregulation of P62 immunoexpression.
Our findings revealed that CsA administration significantly (p < 0.001) deteriorated the renal function markers creatinine and BUN, which is in agreement with the studies by Haleagrahara et al. [41] and Helmy et al. [42]. This can be regarded as the reduction of the glomerular filtration rate or as tubular damage resulting from cyclosporine administration [43,44]. This renal impairment appeared as diffuse cortical cell necrosis with large intraluminal sloughed tubular epithelial cells and necrotic debris (granular casts). These histological alternations were in line with previous studies by Yoon [45] and Lee, [46].
The current study indicated that the subcutaneous injection of CsA significantly (p < 0.001) downregulated the antioxidant enzymes CAT, GPx, and SOD and enhanced MAD as a lipid peroxidation marker. These results are consistent with previous work performed by Capasso et al. [47]. CsA-induced oxidative stress due to the overproduction of oxygen free radicals consumes the protective endogenous antioxidant enzymes, resulting in cellular lipid, DNA, and protein damage [48,49]. MDA elevation results in cellular membrane damage, mainly in the lipid membrane [50]. CsA-induced histological alternation

Discussion
Cyclosporine A administration in organ transplantation is mandatory owing to its immunosuppressive properties, but its prolonged usage causes many side effects, one of which is oxidative-stress-induced nephrotoxicity [34][35][36]. It has been reported that antioxidant intake positively protects individuals against oxidative-insult-induced nephrotoxicity [34]. The antioxidant power of ZW is highly regarded for its alkaline ionized nature [20,37,38]. Analyses of ZW have revealed its high amounts of beneficial trace elements such as zinc, Mg, and selenium, which give ZW its antioxidant and antiapoptotic character [39,40]. Therefore, this study investigated the possible protective effect of ZW against CsA-induced nephrotoxicity along with its underlying mechanisms. From our findings, we can conclude that ZW improves renal impairment induced by CsA administration by decreasing creatinine and BUN levels and elevating creatinine clearance. Moreover, drinking ZW combats CsA-induced oxidative stress by decreasing MDA, a lipid oxidation marker, and enhancing the activities of the antioxidant enzymes SOD, GPx, and CAT, resulting in a marked improvement in the renal architecture. Additionally, ZW eliminates renal tubular epithelial cell apoptosis via a decrease in the apoptotic markers procaspase-8, caspase-8, case-9, caspase-3, cytochrome c, and BAX along with the augmentation of antiapoptotic Bcl-2 immunoexpression as well as the upregulation of the autophagy markers Beclin-1, LC3, and Atg5 and the downregulation of P62 immunoexpression.
Our findings revealed that CsA administration significantly (p < 0.001) deteriorated the renal function markers creatinine and BUN, which is in agreement with the studies by Haleagrahara et al. [41] and Helmy et al. [42]. This can be regarded as the reduction of the glomerular filtration rate or as tubular damage resulting from cyclosporine administration [43,44]. This renal impairment appeared as diffuse cortical cell necrosis with large intraluminal sloughed tubular epithelial cells and necrotic debris (granular casts). These histological alternations were in line with previous studies by Yoon [45] and Lee, [46].
The current study indicated that the subcutaneous injection of CsA significantly (p < 0.001) downregulated the antioxidant enzymes CAT, GPx, and SOD and enhanced MAD as a lipid peroxidation marker. These results are consistent with previous work performed by Capasso et al. [47]. CsA-induced oxidative stress due to the overproduction of oxygen free radicals consumes the protective endogenous antioxidant enzymes, resulting in cellular lipid, DNA, and protein damage [48,49]. MDA elevation results in cellular membrane damage, mainly in the lipid membrane [50]. CsA-induced histological alternation can be explained by the accumulation of reactive oxygen species (ROS), which negatively affects the glomerular endothelium [51]. Hyaline casts can be explained by lipid peroxidation, which disrupts cellular membranes and by the outpouring of cytoplasmic contents into the tubular lumen [52].
ZW drinking water significantly (p < 0.001) improved renal function, downregulated the serum levels of creatinine and BUN, and increased creatinine clearance. These results are in accordance with the findings of El Maleky et al. [53], who stated that drinking ZW with an oral gliclazide hypoglycemic drug markedly normalizes the serum BUN, creatinine, and albumin in STZ-induced nephropathy. Furthermore, Abdullah et al. [54] stated that drinking ZW, which is a good example of alkaline water, exhibited remarkable improvements in metabolic acidosis concomitant with chronic renal failure and hemodialysis. This renoprotective effect of ZW can be explained by its antioxidant power. Considering the higher exposure of renal tubular cells to oxidative stress due to their oxygen consumption [55,56], drinking ZW was shown to significantly (p < 0.001) elevate the homogenate endogenous antioxidant enzymes CAT, GPx, and SOD, while additionally reducing the level of the lipid peroxidation marker MDA. This result is in line with previous research conducted by Abdullah et al. [53], Satta et al. [57], and Bamosa et al. [58]. The antioxidant effect of ZW is highly regarded for its ionized nature, and its analysis revealed its high amounts of Zn, Mn, and Se (Table 4), which have high antioxidant power and a remarkable elevation in their antioxidant enzyme levels [53]. The correction of renal functions by ZW via ROS scavenging also explains the improvement in the renal histological architecture. These findings align with the study by Saif et al. [38], who discussed the effect of ZW in response to carbon-tetrachloride-induced liver-architected modifications, and with the findings of El Maleky et al. [53], who reported on the protective effect of ZW in renal histology in STZ-induced diabetic nephropathy. The pathogenesis of CsA-induced nephrotoxicity is considered to involve the activation of several apoptotic cascades in renal tubular epithelial cells [59]. Three pathways are involved in the pathogenesis of the apoptotic process: the death receptor activation extrinsic pathway, mitochondrial stress intrinsic pathway, and ER stress pathway [60]. There is a substantial link between the mitochondrial pathway and oxidative stress-induced apoptosis [61]. In this study, the Western blotting assay showed that ZW significantly (p < 0.001) increased the protein expression of procaspase-8, caspase-8, calpain, caspase-3, and cytochrome c and that it increased the percentage of Annexin-V-labeled apoptotic renal cells. Several pathways are associated with apoptotic cell death via CsA administration, including oxidative stress via the overexpression of the proapoptotic p53 protein resulting from ROS overproduction, as reported by Koppelstaetter et al. [62]. These findings are in agreement with other works by Tu et al. [63] and Øzbay et al. [64], who reported the DNA fragmentation of CsA and its implication in the pathogenesis of chronic organ diseases. Interestingly, drinking ZW significantly antagonized the apoptotic effect of CsA against renal tubular epithelial cells by decreasing caspase-3 expression and improving its effect on the antiapoptotic protein Bcl-2. These findings are in line with those of El Maleky et al. [53], who proved the antiapoptotic effect of the combination of ZW with gliclazide by exploring their capacity to decrease the immunoexpression of renal tubular epithelial cells stained with caspase-3 in STZ-induced nephropathy. We thus conclude that the antiapoptotic power of ZW can be regarded as its antioxidant property.
Several works have explored the effect of oxidative stress on the regulation of autophagy [65]. Several studies have also discussed the protective role of the autophagy process, which serves as a cellular defense mechanism against CsA-induced nephrotoxicity [66]. Autophagy maintains cellular homeostasis by balancing cellular component degradation and production, thus delaying the apoptotic cellular cells [67]. The results of the present work revealed that CsA significantly (p < 0.001) downregulated the protein expression of the autophagic protein Atg5 and the immunoexpression of the autophagic regulator Beclin-1. It also induced the autophagosome membrane formation of the LC3-II puncta with the upregulation of P62, which is considered to be one of the autophagyassociated proteins whose accumulation is related to autophagy inhibition. However, our results are in disagreement with those of Pallet et al. [10], who demonstrated the upregulating effect of CsA on human-cultured renal tubular epithelial cells through an increase in LC3-II protein expression and autophagosome formation with electron microscopic examination. However, our results are consistent with those of Inoue et al. [68] and Jiang et al. [69], who reported that the autophagy process was upregulated in the initial phases of cisplatin-induced severe kidney pain as a protective mechanism against tubular epithelial cell apoptosis. The works of Li et al. [70] and Ozkok et al. [71] also confirm the previous explanation with their investigations, which showed that the treatment of NRK-52E cells with 20 mmol/L cisplatin markedly upregulated LC3II in the first 6 h of the experiment; however, after 24 h, LC3II markedly decreased to basal level. On the other hand, Zamzam water exerted an upregulating effect on the autophagy process by upregulating the protein expression of Atg5 and the immunoexpression of LC3 and beclin-1 and by decreasing the immunoexpression of P62. This increased the autophagic flux in comparison to the CsA group and indicated that drinking ZW prolongs the procaspase lag period and the subsequent inhibition of renal tubular epithelial cell apoptosis, thus mitigating CsA-induced nephrotoxicity. In the current research, at the mRNA level and with the determination of protein expression using Western blotting, cyclosporine A was shown to significantly (p < 0.001) decrease the phosphorylation of AMPK and increase mTOR phosphorylation. Moreover, Western blotting revealed the decreased protein expression of ULK-1, an autophagy initiator. The downregulating effect of CsA on the autophagy process was most probably due to its impact on the AMPK/mTOR pathway. This result is in disagreement with the findings reported by Park et al. [72], who reported that the intraperitoneal injection of cyclosporine at a dose of 50 mg/kg increased the AMPK level in a rat's hippocampus 5 h after injection. This can be explained as CsA's upregulating effect on the autophagy process at the acute stage. Several studies have demonstrated that the phosphorylation of AMPK potentiates the autophagy process by activating the ULK-1 autophagy initiator kinase under starvation circumstances. In contrast, mTOR phosphorylation inhibits autophagy processes under normal conditions by interfering with the interaction between AMPK and ULK-1 [73]. Recent research has demonstrated that the activation of the AMPK signal has an ameliorative effect in response to acute kidney injury [74]. Drinking ZW was shown to activate the autophagy process in response to CsA-induced renal tubular apoptosis through its modulation of the AMPK/mTOR pathway. Autophagy-apoptosis cellular death is considered to be complex pathway [7,8]. There are three different types of interactions between autophagy and apoptosis which illustrate their crosstalk: First, autophagy collaborates with apoptosis; second, autophagic cellular death antagonizes apoptotic cellular death; third, autophagy enables apoptotic cellular death. The findings of this research showed that CsA injection could upregulate proapoptotic Bax protein immunoexpression and downregulate antiapoptotic Bcl-2 protein immunoexpression, which resulted in the DNA fragmentation of the renal tubular epithelial cell and its subsequent apoptosis. The transfer of Bcl-2 from the cytoplasm to the mitochondria plays a crucial role in propagating apoptotic signals to the cytoplasm [75,76]. On the contrary, the level of Bcl-2 was upregulated during autophagic cellular death, indicating that the autophagy process protects the cells from apoptotic death. It has been demonstrated, both in vivo and in vitro, that the autophagy process is mandatory for cell survival during starvation [77,78], which also protects epithelial cells from the apoptotic pathways [79] during metabolic stress [80]. Based on the aforementioned results, the activation of the autophagy process by drinking Zamzam water offers a unique chance for renal tubular epithelial cell survival in the face of cyclosporine-induced cellular apoptosis and nephrotoxicity.

Conclusions
The present study demonstrated that receiving ZW with the SC injection of CsA ameliorated its nephrotoxicity, as evidenced by the restoration of the renal function parameters and the subsequent improvement of the histological architecture. Additionally, ZW eliminated CsA-induced renal tubular apoptosis by decreasing the protein level of apoptotic markers and increasing the level of the antiapoptotic protein Bcl-2. Moreover, it enhanced the autophagy process by increasing LC3, Becline-1, and Atg5 while decreasing the P62 autophagic marker proteins, with a modulation of the AMPK/mTOR/ULK-1 pathway, thus restraining renal tubular cell apoptosis by boosting the autophagic process.

Data Availability Statement:
The data that support this research will be shared upon reasonable request to the corresponding authors.